Artificial kidney precursor and process for production thereof

ABSTRACT

The present invention provides an artificial kidney precursor containing a non-human mammalian metanephros separated out from a living body, wherein the metanephros has been subjected to freezing and thawing treatments outside a living body, and contains mammalian mesenchymal stem cells transferred outside a living body, and a method of production thereof.

TECHNICAL FIELD

The present invention relates to an artificial kidney precursor and amethod of production thereof.

BACKGROUND ART

The kidney is the major erythropoietin-producing organ; decreasederythropoietin production accompanying renal failure causes renal anemiaas a complication.

In recent years, as next-generation therapeutic methods for renal anemiaas such, methods have been proposed wherein an artificial kidneypossessing a potential for erythropoietin production or a precursorthereof is produced by regenerative medical technology, and this istransplanted to renal anemia patients. For example, Patent Document 1discloses a method for producing erythropoietin-producing organoid(artificial kidney) precursor comprising the step of transplantingisolated mesenchymal stem cell derived from a mammal into an embryowithin a pregnant mammalian host or an embryo separated from a pregnantmammalian host to thereby induce the differentiation of the mesenchymalstem cell, wherein a site to which the mesenchymal stem cell is to betransplanted is a nephrogenic site of the embryo, and a timing oftransplantation corresponds to the stage in which a immune system of thehost is still immunologically tolerant.

In this method, however, there is a problem of complicated processbecause it is necessary to collect amniotic fluid from a pregnant mammaljust before the artificial kidney precursor is transplanted to thepatient, and confirm the biological safety of the precursor.Additionally, mesenchymal stem cells must be injected into anappropriate site in the embryo, that can become the kidney in thefuture; this operation requires high skills. Additionally, becausemesenchymal stem cells must be injected into the embryo while the host'simmune system is in a state of immune tolerance, the flexibility of theentire schedule of treatment is low. In this method, it is difficult toobtain a functional artificial kidney precursor unless whole embryoculture is performed; there arises a risk of contamination due tocultivation, and complicated process of purification after cultivationis necessary.

Hence, there is a demand for the development of a method of producing anartificial kidney precursor wherein 1) the biological safety of theartificial kidney precursor can easily be tested before transplantationto the patient, 2) the operation is simple, 3) flexibility of the entireschedule of treatment can be secured, and 4) no operation of cultivationis required.

[Prior Art Documents] [Patent Document] Patent Document 1: WO2008/004598 SUMMARY OF THE INVENTION Technical Problem

It is an object of the present invention to provide a method ofproducing an artificial kidney precursor wherein 1) the biologicalsafety of the artificial kidney precursor can easily be tested beforetransplantation to the patient, 2) the operation is simple, 3)flexibility of the entire schedule of treatment can be secured, and 4)no operation of cultivation is required.

Solution to Problem

The present inventors conducted extensive investigations to accomplishthe above-described object; as a result, the inventors found that theabove-described problems can be solved by using an artificial kidneyprecursor obtained by freezing and thawing a metanephros separated froman embryo, and transferring patient-derived mesenchymal stem cells intothe thawed metanephros, and developed the present invention.

Accordingly, the present invention relates to the following.

[1] An artificial kidney precursor comprising a non-human mammalianmetanephros separated out from a living body, wherein the metanephroshas been subjected to freezing and thawing treatments outside a livingbody, and comprises mammalian mesenchymal stem cells transferred outsidea living body.[2] The artificial kidney precursor described in [1], wherein themetanephros is of a mammalian embryo in a stage where the expression ofMHC is still immature.[3] The artificial kidney precursor described in [1], wherein theprecursor is capable of acquiring a potential for erythropoietinproduction when transplanted into an omentum of a mammal.[4] A method of producing an artificial kidney precursor, comprising thesteps of:(I) freezing a mammalian metanephros separated out from a living body;(II) thawing the frozen mammalian metanephros; and(III) transferring mammalian mesenchymal stem cells into the metanephrosoutside a living body before the freezing (I) or after the thawing (II).[5] The method described in [4], wherein the metanephros used in (I) isa metanephros separated out from a mammalian embryo in a stage where theexpression of MHC is still immature.[6] The method described in [4], wherein the artificial kidney precursoris capable of acquiring a potential for erythropoietin production whentransplanted into an omentum of a mammal.

Effect of the Invention

Using the method of the present invention, the biological safety of anartificial kidney precursor can easily be tested before transplantation.Using the method of the present invention, it is possible to produce anartificial kidney precursor by simple operations. Using the method ofthe present invention, the entire schedule of treatment can be flexiblydesigned because a precursor of artificial kidney can be produced withdesired timing by using a freeze-preserved metanephros. Furthermore,because the method of the present invention substantially obviates theneed of an operation of cultivation, it involves no risk ofcontamination and does not need a complicated process of purificationafter cultivation.

DESCRIPTION OF EMBODIMENTS

The present invention provides an artificial kidney precursor comprisinga mammalian metanephros separated out from a living body, wherein themetanephros has been subjected to freezing and thawing treatmentsoutside a living body, and comprises mammalian mesenchymal stem cellstransferred outside a living body.

In the present invention, an “artificial kidney” refers to a cellularorganization, not a single cell, having a potential for erythropoietinproduction, which is a function the kidney possesses in nature. The“artificial kidney” in the present invention may not have the functionof filtering waste materials and water in the blood and dischargingurine. Each cell of the artificial kidney contacts with each otherthree-dimensionally except on its surface, the adjoining cellsexchanging information via various intercellular junction, and can havea potential for erythropoietin production regulation.

A “precursor” refers to a tissue that turns into an artificial kidneywhen placed in a living body. Although the “precursor” has factorsnecessary for potentials for erythropoietin production and productionregulation, it is in a state where the potentials for erythropoietinproduction and production regulation are not exhibited because of anenvironmental factor. The artificial kidney precursor is capable ofacquiring a potential for erythropoietin production when transplanted tothe omentum of a mammal.

A “potential for erythropoietin production regulation” means thecapability of producing a large amount of erythropoietin (an amountrequired for amelioration of anemia) at times when a larger amount oferythropoietin than in a normal state is required in the body of therecipient (patient) (in anemia and the like), and producing an amount oferythropoietin required for retaining a normal state in the body attimes when the recipient (patient) is in a normal state (anemia has beenameliorated and the like). That is, the artificial kidney of the presentinvention is advantageous over conventional erythropoietin-producingcells in that it is capable of producing an amount of erythropoietinrequired for the recipient (patient), and capable of producingerythropoietin continuously for a long time by taking necessarynutrients from blood vessels in the omentum.

In the present invention, a “metanephros” refers to a site correspondingto the genesis of the kidney in the mammalian embryo. The metanephrosused in the present invention is preferably such that blast cells of thekidney are in a budding state before the start of development. Themetanephros is located around the ureteric bud budding site in themammalian embryo, more specifically between the somite and lateralplate. The metanephros is preferably the metanephrogenic mesoderm.

As examples of the mammal from which the metanephros is derived,laboratory animals such as rodents such as mice, rats, hamsters andguinea pigs, and rabbits; domestic animals such as pigs, bovines, goat,horses, sheep and minks; companion animals such as dogs and cats;primates such as humans, monkeys, rhesuses, marmosets, orangutans andchimpanzees; and the like can be mentioned. The mammal is preferably arat or pig.

The metanephros is separated from a mammalian embryo outside the livingbody. As the embryo, one in which the expression of MHC (majorhistocompatibility complex) is still immature is suitably used. By usinga metanephros in this stage, immune reactions accompanied bytransplantation of an artificial kidney precursor to the recipient canbe avoided. When the embryo is of a non-primate mammal, it is preferablethat the metanephros be separated out in a stage before an exogenousantigen (carbohydrate antigen such as alpha-Gal) emerges. For example,in experiments using rats, embryo of normally E (stage embryonic day) 9to 16, preferably E10 to 15, more preferably E11 to 14, is used. Forother mammals as well, embryo in equivalent stages can suitably be used.However, preceding or following stages can also be applied, providedthat conditions are chosen. Separation of a metanephros from an embryocan be performed using a stereomicroscope and the like.

Freezing treatment of a metanephros is performed in a cryopreservationliquid conventionally used to freeze mammalian tissue or cells. Thecryopreservation liquid generally contains a cryopreservative such asDMSO, glycerin and the like. The concentration of the cryopreservativeis normally in the range of 5 to 30% by weight, preferably 10 to 20% byweight. The preservation liquid may contain serum. The concentration ofthe serum is normally 5 to 50% by weight, preferably 10 to 30% byweight. As preferred cryopreservation liquids, the ET-KYOTO solutioncontaining DMSO and FCS, CELLBANKER (registered trademark) and the likecan be mentioned.

By freezing the metanephros, immune reactions accompanied bytransplantation of the artificial kidney precursor to the recipient(patient) can be suppressed.

The frozen metanephros can be preserved in a frozen state nearlypermanently, and can be used after being thawed and roused from thesleep as required. Temperature during cryopreservation is generally −80to −200° C., preferably −196° C. (in liquid nitrogen). Because anartificial kidney precursor can be produced at desired timing due tocryopreservation of a metanephros, the entire schedule of treatment canbe flexibly designed.

The frozen metanephros is subjected to thawing treatment in aphysiological culture medium according to a conventional method. Themethod of thawing is not particularly limited; for example, with afreezing tube floating in a 37° C. thermoregulated bath, the frozenmetanephros is thawed by adding dropwise the physiological culturemedium thereto. For eliminating contamination of the cryopreservationliquid, the thawed metanephros is preferably washed with thephysiological culture broth.

The metanephros used in the artificial kidney precursor of the presentinvention comprises mammalian mesenchymal stem cells transferred outsidea living body.

“Mesenchymal stem cells” broadly mean a population of stem cells thatproliferate in the undifferentiated state, and are capable ofdifferentiating into all or some of osteoblasts, chondroblasts,lipoblasts and the like, or progenitor cells thereof. The mesenchymalstem cells used in the present invention possess a potential fordifferentiating into erythropoietin-producing cells of the kidney.

As examples of the mammal from which mesenchymal stem cells are derived,laboratory animals such as rodents such as mice, rats, hamsters andguinea pigs, and rabbits; domestic animals such as pigs, bovines, goat,horses, sheep and minks; companion animals such as dogs and cats;primates such as humans, monkeys, rhesuses, marmosets, orangutans andchimpanzees; and the like can be mentioned. The mammal is preferably arat or human.

The mammal from which the mesenchymal stem cells are derived ispreferably the same animal species as the recipient to whichtransplantation of the artificial kidney precursor of the presentinvention is intended. Most preferably, the recipient's own mesenchymalstem cells are used.

Mesenchymal stem cells can be collected from mammalian bone marrowfluid, peripheral blood, umbilical blood and the like by a publiclyknown ordinary method. For example, human mesenchymal stem cells can beisolated via cultivation and passage of hematopoietic stem cells and thelike obtained by bone marrow aspiration (Journal of Autoimmunity, 30(2008) 163-171).

Mesenchymal stem cells isolated from a living body can also be expandedin vitro while maintaining the differentiating potential thereof bycarrying out adhesion culture in an appropriate medium. The mesenchymalstem cells thus expanded in vitro also fall within the scope of thepresent invention. As the medium, for example, DMEM, EMEM, RPMI-1640,F-12, α-MEM, MSC growing medium (Bio Whittaker) and the like are used.Culturing temperature is generally in the range of about 30 to 40° C.,and is preferably about 37° C. The CO₂ concentration is generally in therange of about 1 to 10%, and is preferably about 5%. Humidity isgenerally in the range of about 70 to 100%, preferably about 95 to 100%.For maintaining a high differentiating potential, it is preferable thatthe cultivation do not extend over 2 to 5 passages or more.

Transfer of mesenchymal stem cells into a metanephros is performedbefore freezing, or after thawing the metanephros. For example,mesenchymal stem cells are injected into a metanephros using amicropipette and the like under a stereomicroscope. The number of cellstransferred is determined as appropriate on the basis of the size of themetanephros and the like; generally about 10⁴ to 10⁶ (for example,5×10⁴) mesenchymal stem cells per rat metanephros are injected.

Although mesenchymal stem cells suspended in a physiological culturemedium may be used for the transfer, mesenchymal stem cells enclosed ina gel containing an extracellular matrix component (laminin, collagentype IV, entactin, heparan sulfate proteoglycan, Matrigel (registeredtrademark) and the like) are suitably used from the viewpoint ofprevention of leakage of intracellular fluid at the time of paracentesisafter cryopreservation.

The size of the artificial kidney precursor of the present inventionneed not to be close to the size of the kidney, which is anerythropoietin-producing organ; a size 1/50 to 1/10 of that of theentire kidney is sufficient.

The artificial kidney precursor of the present invention can be producedby performing the following process:

(I) freezing a mammalian metanephros separated out from a living body;(II) thawing the frozen mammalian metanephros; and(III) transferring mammalian mesenchymal stem cells into the metanephrosoutside a living body before the freezing (I) or after the thawing (II).

The definitions of the respective terms and details of the operationsare as described above.

Although the artificial kidney precursor of the present inventionobtained by the above-described process may be subjected to organculture, it is preferable that organ culture be not performed becausethere is no risk of contamination, and a complicated process ofpurification after cultivation is unnecessary. The artificial kidneyprecursor of the present invention is advantageous in that it is capableof acquiring adequate potentials for erythropoietin production andproduction regulation, when transplanted into a mammal, without beingsubjected to such a process of organ culture. The organ culture can beperformed by placing the artificial kidney precursor on a filter, addingan appropriate medium to a dish thereunder, and allowing the dish tostand in an incubator.

When the artificial kidney precursor of the present invention istransplanted into a living body of a recipient mammal, the precursorengrafts in the recipient's body, and the mesenchymal stem cellscontained in the precursor proliferate and differentiate intoerythropoietin-producing cells of the kidney. As a result, theartificial kidney precursor differentiates into an artificial kidneypossessing potentials for erythropoietin production and productionregulation, which is now capable of producing erythropoietin in theliving body continuously for a long time by taking necessary nutrientsfrom blood vessels. Therefore, the artificial kidney precursor of thepresent invention is useful for the treatment of erythropoietin-relateddiseases. By transplanting the artificial kidney precursor of thepresent invention to a mammal, erythropoietin-related diseases in themammal can be treated.

In the present invention, an “erythropoietin-related disease” is adisease associated with a reduction of the amount of erythropoietinproduced, including anemias related to renal failure, diabetes, ulcers,cancers, infections, dialysis, surgery or chemotherapies. In particular,in case of anemia due to chronic renal failure, erythropoietin cannot beproduced because of progressive destruction of renal parenchyma andliver function, so the erythropoietin concentration in the circulationdoes not rise; this is a major problem.

As examples of the recipient mammal, laboratory animals such as rodentssuch as mice, rats, hamsters and guinea pigs, and rabbits; domesticanimals such as pigs, bovines, goat, horses, sheep and minks; companionanimals such as dogs and cats; primates such as humans, monkeys,rhesuses, marmosets, orangutans and chimpanzees; and the like can bementioned. The recipient mammal is preferably a rat or human.

Although the method of transplanting the artificial kidney precursor ofthe present invention into a mammal is not particularly limited, as faras the precursor can engraft in the recipient's body and acquirepotentials for erythropoietin production and production regulation, theartificial kidney precursor is preferably transplanted into therecipient mammal's omentum. Transplantation into the omentum can beperformed by an ordinary surgical procedure; for example, a methodcomprising picking up the tissue to be transplanted with a sharpforceps, making a small incision on the surface of the adipose tissue inthe omentum with the forceps tip, and embedding the tissue in theincision, can be mentioned. The artificial kidney precursor of thepresent invention can also be transplanted into the omentum by means ofan endoscope.

The dose of the artificial kidney precursor of the present inventionvaries depending on the subject of administration, target disease,symptoms and the like; generally, an artificial kidney precursorprepared from a swine metanephros is transplanted into an adult humanrenal anemia patient (a 60 kg body weight assumed), it is convenient totransplant about 10 metanephroses in one time of surgery, and toincrease the number of metanephroses step by step while monitoring theseverity of the patient's anemia, if required. In case of other animals,an amount calculated per 60 kg body weight can be transplanted.

The present invention is hereinafter described more specifically bymeans of the following Example, to which, however, the invention isnever limited in any way.

EXAMPLE

Metanephroses were separated from rat embryos at day 14 of gestation,and then cryopreserved in ET-Kyoto+10% DMSO+20% FCS. The diameters wereabout 2 mm.

After cryopreservation for 2 days, the frozen metanephroses were thawed.Rat mesenchymal stem cells were enclosed in Matrigel, and injected intothe metanephroses at 1×10⁴ cells per metanephros. Thereafter, themetanephroses were transplanted to the omenta of syngenic rats.

Seven days after the transplantation, the grown metanephroses wereseparated from the rats having received the metanephros transplants. Thenumber of animals having received the metanephros transplants was 2, ineach of which three and six, respectively, out of seven metanephroses,grew. The metanephroses grew to diameters of 6 to 8 mm. Erythropoietinproduction in the grown artificial kidney precursors was confirmed bythe same method as that described in Transplantation VOL 85, No 11, Jun.15, 2008, i.e., immunostaining and genetic analysis oferythropoietin-producing cells.

INDUSTRIAL APPLICABILITY

Using the method of the present invention, the biological safety of anartificial kidney precursor can easily be tested before transplantation.Using the method of the present invention, it is possible to produce anartificial kidney precursor by simple operations. Using the method ofthe present invention, the entire schedule of treatment can be flexiblydesigned because a precursor of artificial kidney can be produced withdesired timing by using a freeze-preserved metanephros. Furthermore,because the method of the present invention substantially obviates theneed of an operation of cultivation, it involves no risk ofcontamination and does not need a complicated process of purificationafter cultivation.

This application is based on a patent application No. 2008-173935 filedin Japan (filing date: Jul. 2, 2008), the contents of which areincorporated in full herein by this reference.

1. An artificial kidney precursor comprising a non-human mammalianmetanephros separated out from a living body, wherein the metanephroshas been subjected to freezing and thawing treatments outside a livingbody, and comprises mammalian mesenchymal stem cells transferred outsidea living body.
 2. The artificial kidney precursor according to claim 1,wherein the metanephros is of a mammalian embryo in a stage where theexpression of MHC is still immature.
 3. The artificial kidney precursoraccording to claim 1, wherein the precursor is capable of acquiring apotential for erythropoietin production when transplanted into anomentum of a mammal.
 4. A method of producing an artificial kidneyprecursor, comprising the steps of: (I) freezing a mammalian metanephrosseparated out from a living body; (II) thawing the frozen mammalianmetanephros; and (III) transferring mammalian mesenchymal stem cellsinto the metanephros outside a living body before the freezing (I) orafter the thawing (II).
 5. The method according to claim 4, wherein themetanephros used in (I) is a metanephros separated out from a mammalianembryo in a stage where the expression of MHC is still immature.
 6. Themethod according to claim 4, wherein the artificial kidney precursor iscapable of acquiring a potential for erythropoietin production whentransplanted into an omentum of a mammal.